Dysfunctional regulation of sodium-iodide symporter (NIS) trafficking can result in ineffective radioiodide uptake in patients with differentiated thyroid cancer. Understanding the trafficking pathways of this key protein can be used to optimise radioiodide therapy. Recently, we identified via HiLo microscopy that the protein ADP-ribosylation Factor 4 (ARF4) helps shuttle NIS to the plasma membrane. To understand how ARF4 interacts with NIS mechanistically, we utilised advanced imaging techniques, primarily using Zeiss 780 confocal microscopy to generate 3D images of NIS-GFP and ARF4-mCherry expressing cells. This allowed us to investigate the location of interaction and how this may be manipulated to maximise trafficking towards the plasma membrane. Targeted mutagenesis of the small GTPase ARF4 indicated that the interaction between NIS and ARF4 occurs independently of guanine-nucleotide binding status. Two compounds reported to induce ARF4 expression, phorbol 12-myristate 13-acetate (PMA) and γ-tocotrienol, were tested for their capacity to upregulate ARF4 expression, but neither compound significantly altered ARF4 function in thyroid TPC1 and breast MDA-MB-231 cell lines. Analysis of data from The Cancer Genome Atlas revealed that lower ARF4 expression significantly correlates with positive BRAF mutation status in papillary thyroid tumours. Tetracycline inducible cell lines expressing myc-BRAF were therefore used to investigate whether BRAF is able to regulate ARF4 function. However, ARF4 expression was not affected following induction of BRAF protein levels over time, nor by the mutational status of BRAF, suggesting the MAPK pathway is not a direct regulator of ARF4 expression. Further elaboration of the post-translational pathways involved in shuttling NIS to the plasma membrane is now required to discern how these may be targeted therapeutically to optimise iodide uptake for patients with therapeutically insufficient levels of radioiodine uptake.